Project Summary Protein binding events are essential to most cell functions and often lead to disease when disrupted. Of particular interest here are very high affinity protein interactions with dissociation constants (Kd) in the femtomolar range. The physical origin of the large binding free energy involved in these interactions is not well understood. In the literature, these extreme affinities have been largely ascribed to enthalpic contributions (structural interactions) and the hydrophobic effect (entropy of water). In preliminary results with barnase:barstar, one of the highest affinity protein-protein interactions known, we found that this view is incomplete. Recently, the Wand laboratory has developed a ?conformational entropy meter? that is able to quantitatively relate measurements of fast (ps-ns) dynamics of methyl-bearing side chains to the conformational entropy. Conceptually, the approach relies on the idea that the motion indirectly reports on the distribution of microstates accessible to the system. Our initial study of the high affinity barnase:barstar complex reveals an unprecedented role for conformational entropy. A widespread increase in fast motions occurs upon formation of the complex, particularly in regions distant from the binding interface. This corresponds to a large and favorable change in conformational entropy upon binding of about -18 kcal/mol. It follows that we should be able to decrease the binding affinity of extremely high affinity complexes by restricting motions in barnase:barstar. To test this, intramolecular disulfide bridges will be introduced in both proteins to rigidify the structures (akin to molecular stapling). The effect on global thermodynamics will be studied by calorimetry. Affinties in the very high regime will be measured by a competitive inhibition kinetics assay. Successful candidates with decreased affinity will be studied using NMR relaxation methods to measure dynamics of the backbone and methyl-bearing sides chains. The ?conformational entropy meter? will be used to interpret quantitatively the changes in motion as changes in T?Sconf. Furthermore, the generality of this approach will be evaluated by using the same strategy to study other extreme affinity complexes, such as the bacterial cognate protein complex E9:Im9 (Kd ~ 10 M). Lastly, the total binding entropy of barnase:barstar -15 has been reported as near-zero. This means that the contribution we find of -18 kcal/mol from T?Sconf must be compensated by a similar but unfavorable contribution from solvent entropy. This is consistent with the ~20 water molecules seen trapped at the interface in the crystal structure. To test whether these waters exist in solution and are truly constrained, reverse micelle technology will be used to trap single proteins with only a few layers of water. Confinement in reverse micelles allows tracking of the protein-water interaction times and will reveal the slowed dynamics of water molecules trapped at the barnase:barstar interface. This work will directly evaluate the role of conformational entropy in the formation of very high affinity protein complexes.